Pyrophosphorolysis-activated polymerization (PAP) is a technique useful for nucleic acid polymerization and amplification. PAP is an allele-specific amplification method, the essence of which is that 3′-dideoxynucleotide-terminated primers (also designated as “P*”) are activated with a specific polymerase enzyme, in the presence of pyrophosphate (also designated as “PPi”). As a result, the P* primer is hybridized with the complementary target sequence.
Accordingly, PAP allows one to obtain a desired nucleic acid strand on a nucleic acid template strand. This is useful for detecting rare mutations with high selectivity. PAP is a promising technology in the field of cancer diagnostics, genotoxicology, amplification with increased specificity, nucleic acid synthesis, and other applications.
Some features of PAP have been described previously. See, e.g., U.S. Pat. No. 7,033,763 to Liu et al., the contents of which are incorporated herein by reference in its entirety. Briefly, the PAP method serially couples pyrophosphorolysis with polymerization by DNA polymerase for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification.
The basic steps of the PAP process involve annealing to a nucleic acid an oligonucleotide P* having a non-extendable 3′ end. The 3′ non-extendable terminus of the oligonucleotide P* is removed by pyrophosphorolysis and an unblocked oligonucleotide can be obtained as a result. Finally, the unblocked oligonucleotide can be extended and the presence of the nucleic acid can be detected by detecting the extended oligonucleotide. The chemistry involved in the process of pyrophosphorolysis is the reaction of pyrophosphate with a 3′-nucleotide monophosphate (NMP) which is removed from duplex DNA.
While PAP is undoubtedly a very attractive technique, it has certain deficiencies and drawbacks. One difficulty lies in the process of obtaining a suitable oligonucleotide P. For instance, such products as ddA, ddG, and dT are not available commercially and have to be synthesized. Previously, the synthesis of those ddN-terminated oligonucleotides P* was conducted by using terminal transferase. Alternatively, 5′-to-3′ reverse synthesis was employed, using 2′,3′-ddA, 2′,3′-ddG or 2′,3′-ddT CE phosphoroamidite, dA-5′, dT-5′, dG-5′, or dC-5′ CE phosphoroamidite, and 5′-dA, dG, dC or dT support. All such synthetic methods are labor intensive and costly.
For dT, the most favorable point of attachment is via the N3 imino nitrogen on the pyrimidine ring, but no N3-protected derivative of 3′-deoxythymidine is available or has been described. Likewise, no adequate method for making a DNA containing dT has been described, other than expensive 5′-to-3′ reverse synthesis. This is likely due to the fact that there is a lack of convenient exocyclic functional group for covalent linkage on the thymine base. Attachment via an exocyclic oxygen, such as 4-oxo, is possible, but alkoxides thus formed are known to be displaced from thymine by nitrogen nucleophiles, such as ammonia, which are used during oligonucleotide deprotection and cleavage. As a result, 3′-terminal thymine can convert to a cytidine derivative.
Accordingly, better methods are needed for preparing oligonucleotides P* suitable for the PAP process. Such methods would allow for easy synthesis of the oligonucleotides and labeled primers, without resorting to expensive 5′-amidite synthesis or to 5′ to 3′ reverse synthesis. The primers so produced would have all the beneficial properties of the primers obtainable by the above-described earlier methods, such as selectivity of PAP in allele-specific PCR. The instant specification describes such synthetic methods.